A Defective Response to Hedgehog Signaling in Disorders of Cholesterol Biosynthesis

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A defective response to Hedgehog signaling in disorders of cholesterol biosynthesis

Michael K. Cooper1,2,5, Christopher A. Wassif3, Patrycja A. Krakowiak3, Jussi Taipale1, Ruoyu Gong1, Richard I. Kelley4, Forbes D. Porter3 & Philip A. Beachy1

Published online 24 March 2003; doi:10.1038/ng1134

Smith–Lemli–Opitz syndrome (SLOS), desmosterolosis and lath- additional role for cholesterol in Hh signal response was sug- osterolosis are human syndromes caused by defects in the final gested by the observation that cyclopamine and jervine, terato- stages of cholesterol biosynthesis. Many of the developmental genic plant alkaloids that block Hh signaling, also inhibit malformations in these syndromes occur in tissues and struc- cholesterol transport and synthesis2,3. But cyclopamine has since tures whose embryonic patterning depends on signaling by the been shown to specifically inhibit Hh signaling by binding to a Hedgehog (Hh) family of secreted proteins. Here we report that pathway component4, and the doses of these alkaloids required response to the Hh signal is compromised in mutant cells from to inhibit Hh signaling are lower than those required to block mouse models of SLOS and lathosterolosis and in normal cells cholesterol transport (ref. 5 and M.K.C., unpublished data). pharmacologically depleted of sterols. We show that decreas- To determine how cholesterol may affect Hh signaling in embry- ing levels of cellular sterols correlate with diminishing respon- onic development, we exposed chick embryos to cyclodextrin, a http://www.nature.com/naturegenetics siveness to the Hh signal. This diminished response occurs at cyclic oligosaccharide that forms non-covalent complexes with sterol levels sufficient for normal autoprocessing of Hh protein, sterols6 and can be used to extract and deplete cholesterol from liv- which requires cholesterol as cofactor and covalent adduct. We ing cells7. Cyclodextrin treatment caused variable loss of the fron- further find that sterol depletion affects the activity of tonasal process and other midline structures (Fig. 2a), and the Smoothened (Smo), an essential component of the Hh signal spectrum of facial defects was similar to that resulting from expo- transduction apparatus. sure to the Hh-pathway antagonist jervine3. The most severely The role of cholesterol in Hh protein biogenesis suggested that affected embryos developed a proboscis-like structure that pheno- impaired Hh autoprocessing might underlie some of the devel- copies the nasal rudiments of mouse embryos that are homozygous opmental abnormalities in SLOS (Fig. 1 and Table 1; ref. 1). An with respect to mutations in the gene Sonic hedgehog (Shh; ref. 8).

Fig. 1 Cholesterol biosynthesis and a b Hh pathway. a, Autosomal recessive HMG CoA disorders with multiple developmen- - SLOS

© Group 2003 Nature Publishing HMG-CoA reductase tal anomalies are associated with lathosterolosis three different enzymatic defects HhNp (represented by solid colored lines) 3 8 desmosterolosis 4 in the final steps of the cholesterol HO 7 lanosterol 6 biosynthesis pathway. SLOS results Ptc Smo from defects in DHCR7 (red line), palmitate lathosterolosis from defects in SC5D (green line) and desmosterolosis from 24 cholest-7,24- lathosterol defects in 3-β-hydroxysterol-∆ - HO HO en-3β-ol cholesterol reductase (yellow line). Steps in sterol synthesis not shown are indicated by Gli proteins multiple arrows. b, After cleavage of Hh precursor protein the signal sequence (black box), the Hh precursor undergoes an internal 7-dehydro- 7-dehydro- HO HO cleavage reaction mediated by desmosterol cholesterol sequences in the C-terminal autopro- Ptch cessing domain (white box). Choles- terol participates in the reaction and nucleus nucleus remains esteriﬁed to the newly desmosterol cholesterol Hh-generating cell Hh-responding cell formed C terminus of the signaling HO HO domain (shaded box). Fully processed, secreted Hh proteins (designated HhNp, p for processed) also receive an N-terminal palmitoyl group in a reaction requiring the acyltransferase Skinny hedgehog30. The response to Hh is regulated by two transmembrane proteins, Ptch (in red) and Smo (in green). Ptch suppresses the activity of Smo, and Hh binding to Ptch inhibits this function (–), leading to Smo activation of a transcriptional response through the Gli family of transcription factors. Ptch is a transcriptional target of Hh signaling and thus forms a neg- ative feedback loop that ensures adequate regulation of Smo in the absence of Hh. Multiple arrows indicate the participation of a complex of Hh signaling components not shown.

Departments of 1Molecular Biology and Genetics and 2Neurology, Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA. 3Heritable Disorders Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892-1830, USA. 4Kennedy Krieger Institute, Baltimore, Maryland 21205, USA. 5Present address: Department of Neurology, Vanderbilt University Medical Center, Nashville, Tennessee 37232, USA. Correspondence should be addressed to P.A.B. (e-mail: [email protected]).

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Table 1 • Developmental malformations in tissues patterned by Hh signaling

Hh family Source of Target Normal role of Consequence of member Hh signal tissue Hh signaling disrupted Hh signaling PHS25 SLOS25 Lath26 Des27

Prechordal plate Ventral Formation of Holoprosencephaly + + – + mesendoderm neuroectoderm midline forebrain and ectoderm and mesenchyme and facial of facial processes of facial processes structures Sonic Posterior limb bud Limb bud Patterning of the Post-axial + + + hedgehog mesenchyme mesenchyme autopod and polydactyly and + + + and ectoderm zeugopod syndactyly Lung bud Lung bud Branching Unilobular + + epithelium mesoderm morphogenesis lungs of lung Prehypertrophic Chondrocytes Regulation of Rhizomelia + + chondrocytes and osteoblasts endochondral Indian bone growth hedgehog Colonic Neural crest Development of Aganglionic + + epithelium cells (?) the enteric megacolon nervous system (Hirschsprung disease) Desert Sertoli cells Leydig cells Development of the Cryptorchidism/ +/+ +/+ +/+ +/– hedgehog male gonad ambiguous genitalia or hypospadias Overlap of developmental anomalies in tissues patterned by members of the mammalian family of Hh signaling proteins28 among individuals with Pallister–Hall syndrome (PHS), SLOS, lathosterolosis (Lath) and desmosterolosis (Des). PHS is an autosomal dominant disorder associated with mutations in GLI3 that reduce transcriptional activation of Hh pathway targets29.

http://www.nature.com/naturegenetics We further investigated Shh signaling in embryonic tissues by HNF3β (hepatocyte nuclear factor 3β or forkhead box A2; ref. 3), exposing chick neural-plate explants to recombinant Shh protein an indicator of ﬂoor plate fate. This high-threshold response was (ShhN, 30 nM) in the presence of cyclodextrin (Fig. 2b). The blocked in the presence of 400 µM cyclodextrin and replaced by response to this level of ShhN protein was uniform production of nearly uniform expression of ISL1, an intermediate-threshold

a definitive embryonic streak stage day 5 Fig. 2 Cyclodextrin treatment inhibited Shh signaling. a, CD 375 µM Cyclodextrin treatment of chick embryos in ovo caused holoprosencephaly. Scanning electron micrographs of facial experimental timeline features at embryonic day 5 of a control (control) chick embryo and of embryos exposed to 375 µM cyclodextrin lnp * (CD). Cyclodextrin treatment at the early-primitive-streak * * stage led to variable loss of midline tissues, primarily the fnp * frontonasal process (fnp), and approximation or fusion of opt mx paired lateral structures, such as the lateral nasal processes

© Group 2003 Nature Publishing mn (lnp), optic vesicles (opt) and the maxillary (mx) and control CD CD CD mandibular (mn) processes. The slanting nostrils of less severely affected chick embryos treated with cyclodextrin resembled the anteverted nares characteristic of SLOS. Com- b ShhN intermediate neural plate explant assay plete loss of the frontonasal process and subsequent fusion CD of the lateral nasal processes (marked by the asterisk at the 48 h experimental timeline top border of the lateral nasal process) led to the formation of a proboscis with a single nasal pit, positioned above fused optic vesicles. b, Cyclodextrin treatment inhibited the cellular response to Shh protein. Confocal microscope images of HNF3β stage 9–10 chick embryo ectoderm dissected from a region of the neural plate intermediate to the notochord and roof plate and just rostral to Hensen’s node. Explants were cultured for 48 h in collagen and stained for HNF3β (red) or ISL1 (green). Neural progenitor cells explanted from this position and at this developmental stage did not express markers of either ﬂoor-plate (HNF3β) or motor-neuron (ISL1) cell fates. Addition of 30 nM ShhN at the onset of cul- ISL1 ture induced a high-threshold response indicated by the expression of HNF3β. In the presence of 400 µM cyclodextrin, however, HNF3β induction was completely blocked. ISL1 expression is indicative of an intermediate-threshold ShhN (nM) 0 30 30 30 response and was not present with 30 nM ShhN. But, in the CD (µM) 0 0 200 400 presence of 200 and 400 µM cyclodextrin, partial inhibition of the high-threshold signaling dose of 30 nM ShhN pro- dorsal neural plate and epidermal ectoderm explant duced the intermediate-threshold response of ISL1 expres- c CD 400 µM sion. c, Cyclodextrin treatment did not inhibit a BMP- 48 h mediated signaling event. Dorsal neural plate progenitor experimental timeline cells and an endogenous source of BMP, the adjacent epidermal ectoderm, were dissected from a region just rostral to Hensen’s node in stage 9–10 chick embryos. After 48 h of culture in the presence of 400µM cyclodextrin, phase contrast and confocal microscope images show that neural crest–like, HNK1-positive cells migrated from the explant into the surrounding collagen. 6 h 48 h 48 h HNK1

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Fig. 3 Cells defective in cholesterol assay biosynthesis did not respond to Shh. a a, Shh autoprocessing proceeded to +/– CD –/– –/– completion in Dhcr7 and Sc5d 30 min 36 h experimental timeline MEFs at low cholesterol levels. Embry- onic fibroblasts generated from Dhcr7 +/– Dhcr7 –/– Sc5d +/– Sc5d –/– Dhcr7+/– (lanes 4–6), Dhcr7–/– (lanes 7–9), Sc5d+/– (lanes 13–15) and Sc5d–/– (lanes 16–18) mice were transiently transfected with a full-length Shh kDa CD/ LDS FBS LDS CD/ LDS FBS LDS ShhN ShhNp mix 1 + 2 FBS ShhN ShhNp mix 10 + 11 CD/ LDS LDS CD/ LDS FBS LDS expression construct and cultured in Shh 50 full serum (fetal bovine serum, FBS; lanes 4, 7, 13 and 16), lipid-depleted 37 serum (LDS; lanes 5, 8, 14 and 17) or lipid-depleted serum after 30 min of treatment with cyclodextrin (CD/LDS; lanes 6, 9, 15 and 18). Shh was effi- 25 ShhN ciently processed under all culture conditions as there was no detectable accumulation of precursor protein (Mr = 45 kDa). Purified ShhNp (lanes 2 and ShhNp 132465798 1011 12 1314 15 1617 18 11; Mr = 19.5 kDa) was cell-associated and migrated faster than unprocessed ShhN protein (lanes 1 and 10) col- lected from the medium of cultured cells transfected with a construct car- 125 Dhcr7 +/– 150 Sc5d +/– rying an open reading frame trun- Dhcr7 –/– –/– b 100 125 Sc5d cated after Gly198 (both ShhNp and 100 ShhN are loaded in lanes 3 and 12), 75 indicating that ShhNp from the 75 treated MEFs (lanes 4–9) probably car- 50 ried a sterol adduct. b, Response of 50 http://www.nature.com/naturegenetics –/– –/– Dhcr7 and Sc5d MEFs to Shh pro- 25 25 tein was inhibited at low cholesterol relative pathway activity 0 0 levels. The effect of cholesterol deple- lipid- lipid- lipid- lipid- +/– serum full full tion on responsiveness of Dhcr7 depleted depleted depleted depleted (gray bars), Dhcr7–/– (maroon bars), cyclodextrin 3.8 mM ––+ ––+ Sc5d+/– (gray bars) and Sc5d–/– (green cholesterol (µg/mg) 39 29 43 23 30 14 49 26 51 26 40 14 bars) MEFs to Shh signaling was total sterols (µg/mg)444047333929 51 33 53 35 43 29 assayed by transfection with a Hh- responsive firefly luciferase reporter and treatment with purified ShhNp (10 nM). MEFs were transfected at sub-confluent densities, cultured to maximum cell density and then induced with ShhNp either in culture medium containing fetal bovine serum (full) or lipid-depleted serum. Some of the cells cultured in lipid-depleted serum were stripped of surface cholesterol with a 30-min exposure to cyclodextrin just before ShhNp induction. For each culture condition, normalized firefly luciferase activity from control and ShhNp treated cells was used to calculate the relative induction, expressed as a percentage of the Shh induction achieved in MEFs cultured in full serum. Bars represent the standard error from quadruplicate experiments.

© Group 2003 Nature Publishing response indicative of motor-neuron fate. At 200 µM cyclodex- proceeds to completion because cholesterol levels are only trin, both HNF3β and ISL1 were expressed, suggesting that lower reduced by roughly 50% under the conditions used for depletion levels of cyclodextrin were less inhibitory. As active ShhN protein (Fig. 3b) and because 7-dehydrocholesterol and lathosterol both was exogenously supplied, this dose-dependent inhibition sug- participate efficiently as sterol adducts in the Shh processing reac- gests that sterol deficits affect response to the Hh signal. Further- tion3. Likewise, cholesterol levels are reduced but never absent in more, this effect seemed to be specific, as treatment with 400 µM the serum of individuals with SLOS12. cyclodextrin alone did not inhibit BMP-induced migration of In contrast with their normal Shh autoprocessing, MEFs with neural-crest cells9 (Fig. 2c). mutations in Dhcr7 and Sc5d had clear deficiencies in their ability Hh protein biogenesis involves internal cleavage and covalent to respond to Shh signal when transiently treated with cyclodex- addition of cholesterol through an autoprocessing reaction1. trin and grown in lipid-depleted culture medium (Fig. 3b). These Cyclodextrin treatment of cultured cells has previously been results indicate that signal response is more sensitive than is sig- reported to interfere with Shh autoprocessing10, an effect distinct nal biogenesis to perturbations of cholesterol homeostasis. Cells from inhibition of response that we observed. To further investi- heterozygous with respect to the mutations in Dhcr7 and Sc5d gate whether signal production or signal response is the prevail- maintained a normal response to ShhNp stimulation under all ing inhibitory mechanism in cholesterol synthesis disorders, we growth conditions (Fig. 3b), presumably because there was suffi- established embryonic fibroblast cell lines from mouse models of cient synthetic activity from the functional allele. In these experi- SLOS11 and lathosterolosis and examined Shh signal biogenesis ments, the initial transient cyclodextrin treatment is needed to and response in parallel under identical culture conditions. reduce sterol levels to below 40 µg mg–1 protein to affect pathway Shh protein was efficiently processed in mouse embryonic response (Fig. 3b). fibroblasts (MEFs) lacking 7-dehydrocholesterol reductase To further investigate Hh signal response in other cultured (Dhcr7) or lathosterol 5-desaturase (Sc5d) enzymes (models of cells, we tested the ability of pharmacological interventions to SLOS and lathosterolosis, respectively; see Fig. 1b), with no mimic the effects of genetic defects in sterol biosynthesis. Con- observable effect of transient cyclodextrin treatment or growth in tinuous treatment with 500 µM cyclodextrin produced about lipid-depleted culture medium (Fig. 3a). All of the processed N- 50% inhibition of Shh signal response in NIH3T3 cells, and terminal product (ShhNp) from mutant cultures was cell-associ- 2 mM cyclodextrin produced nearly complete inhibition ated and had an electrophoretic mobility suggestive of sterol (Fig. 4a). Transient cyclodextrin treatment for 30 minutes before modification (Fig. 3a). The autoprocessing reaction probably exposure to ShhNp, however, did not affect response, even at

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15 mM cyclodextrin, a concentration 30 times higher than the not shown), and the degree of pathway inhibition in sterol biosyn- IC50 for continuous cyclodextrin treatment (Fig. 4b). ShhNp thetic mutant and statin-treated fibroblasts correlated inversely response was also not affected by continuous exposure to com- with total sterol levels (Fig. 4d). An inhibitory role for 7-dehydro- pactin, an inhibitor of 3-hydroxy-3-methyl-glutaryl coenzyme A cholesterol, lathosterol and other cholesterol precursors that accu- (HMG CoA) reductase that blocks sterol synthesis13. The combi- mulate in the Dhcr7 and Sc5d mutant cells seems improbable nation of transient cyclodextrin treatment with continuous com- because synthesis of such precursors in statin-treated wild-type pactin exposure, however, blocked Shh signal response (Fig. 4b). cells is blocked. Indeed, the correlation between responsiveness and These data suggest that compactin treatment, when combined overall sterol content (Fig. 4d) suggests that these precursors may with transient cyclodextrin treatment, can mimic a genetic defect contribute to pathway response, although their effectiveness rela- in sterol biosynthesis to inhibit Shh signal response. The Shh tive to that of cholesterol is unknown. autoprocessing reaction was not affected by these experimental The ability to pharmacologically mimic sterol biosynthetic conditions (data not shown). defects affords an opportunity to identify the Hh pathway com- Cyclodextrins form complexes with hydrophobic compounds ponent most directly affected by sterol perturbations. Patched including proteins and phospholipids in addition to sterols6. But (Ptch) and Smoothened (Smo) stand out as candidate targets the effect of cyclodextrin treatment is probably due to sterol deple- because of their integral membrane structure and essential roles tion, as transient cyclodextrin treatment only inhibited pathway in regulating cellular response to the Hh signal. Genetic and bio- activity in the presence of sterol biosynthetic mutations or of a chemical evidence indicates that Ptch suppresses the activity of statin, and no measurable recovery from the impact of deficits in Smo and that Hh binding to Ptch releases this suppression, molecules other than sterols was achieved during prolonged incu- allowing Smo to activate a transcriptional response through the bation after transient cyclodextrin treatment (Fig. 4c). Furthermore, other sterol-spe- a ShhNp assay b ShhNp assay cific perturbations, such as +/– CD +/– CD +/– compactin 36 h 30 min 36 h treatment with nystatin or fil- experimental timeline experimental timeline http://www.nature.com/naturegenetics 14 ipin , also blocked the response 125 125 of cells to ShhNp protein (data 100 100

75 75 Fig. 4 Inhibition of Hh signal response by sterol depletion in cells with intact 50 50 cholesterol biosynthesis. Hh signal response was inhibited either by 25 25 chronic cyclodextrin treatment (a) or relative pathway activity (%) relative pathway activity (%) 0 0 by statin exposure after acute ShhNp 4 nM + + + + + + ShhNp 4 nM – +++++++ cyclodextrin treatment (b). a, NIH3T3 ﬁbroblasts stably transfected with a cyclopamine 5 µm – + –––– compactin 20 µM +++++++– Hh-responsive luciferase reporter cyclodextrin (µM) ––250 500 1,000 2,000 cyclodextrin (mM) ––0.9 1.9 3.8 7.5 15 15 construct (Shh-LIGHT Z3 cells) were chronically depleted of sterols by the addition of cyclodextrin (CD) to the ShhNp assay assay assay culture medium for the duration of c © Group 2003 Nature Publishing +/– CD compactin ShhNp induction (see schematic in a). Cyclodextrin treatment inhibited the 30 min 36 h 48 h 72 h response to Shh signaling in a dose- experimental timeline dependent manner and to a compa- 125 rable degree as 5 µM cyclopamine16. b, Shh signal reception was also 100 blocked in Shh-LIGHT Z3 cells by acute sterol depletion with transient expo- 75 36 h 48 h 72 h sure to cyclodextrin before ShhNp induction, followed by the addition 50 of an HMG-CoA reductase inhibitor (compactin) during the period of 25 induction (see schematic in b). The

combination of transient cyclodextrin relative pathway activity (%) 0 treatment followed by inhibition of ShhNp 4 nM + + + + + + + + + + + + + + + + + + cholesterol synthesis blocked the compactin 20 µM ++++++++++++++++++ response to ShhNp, whereas neither treatment alone did so. c, Hh path- cyclodextrin (mM) 0 0.9 1.9 3.8 7.5 15 0 0.9 1.9 3.8 7.5 15 0 0.9 1.9 3.8 7.5 15 way inhibition persisted for 72 h after sterols (µg/mg protein) 42 36 32 28 25 22 cyclodextrin treatment. The 3-d recovery period was intended to allow replenishment of non-sterol d 125 cellular components depleted by transient cyclodextrin treatment 100 while maintaining sterol depletion with HMG-CoA reductase inhibition 75 –/– by compactin. d, Total cellular sterols Dhcr7 from parallel cultures of NIH3T3 (c, 36 50 Sc5d –/– h), Dhcr7–/– and Sc5d–/– (Fig. 3b) MEFs NIH3T3 were determined by gas chromatog- 25 raphy–mass spectrometry analysis of extracted lipids and plotted against relative pathway activity (%) 0 the relative Shh pathway activity. Bars 01020304050 represent one standard error (qua- µ druplicate wells). total sterols ( g/mg protein)

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Fig. 5 Sterol depletion inhibited Shh signaling downstream of Ptch at the level a 125 of Smo. a, The constitutively active Shh pathway in Ptch–/– MEFs (measured as β-galactosidase activity produced from a fusion of lacZ to the third codon of 100 Ptch16) was blocked by cholesterol depletion. b, Shh pathway activation by overexpression of wild-type Smo in NIH3T3 cells was also inhibited by choles- 75 terol depletion. But Shh pathway activity driven by overexpression of onco- Ptch–/– genic Smo (c; SmoA1, W539L) was resistant to inhibition by cholesterol 50 depletion. Cyclodextrin and compactin treatments for a–c are as indicated for Figure 3b. NIH3T3 ﬁbroblasts in b and c were stably transfected with expres- 25 sion constructs for either Smo (b) or activated Smo (SmoA1; c), a Gli-luciferase reporter and lacZ for normalization. Forskolin (100 µM) inhibition of SmoA1- driven pathway activity is illustrated as a positive control16. Bars represent one

relative pathway activity (%) 0 compactin 20 µM ++++++– standard error from three (a) or six (b,c) replicates for each treatment group. cyclodextrin (mM) – 0.9 1.9 3.8 7.5 15 15

to cholesterol deprivation of activated Smo suggests that Smo con- formation may be the target of cholesterol deprivation. This effect 150 could be mediated either through direct interaction of cholesterol b with Smo or through an impact on membrane properties, as 125 reported for the function of other seven-transmembrane-domain 100 proteins, such as the oxytocin or the brain cholecystokinin recep- Smo 17 75 tors . One possibility is that sterol depletion could affect a lipid microdomain or raft-mediated process18 required for Smo activity, 50 although we did not observe a change in Smo fractionation with 25 respect to detergent-resistant membranes on sterol depletion (data not shown). Alternatively, the effects of sterol depletion on Smo relative pathway activity (%) 0 compactin 20 µM ++++++– activity might be indirectly mediated through an as yet uncharacter- http://www.nature.com/naturegenetics cyclodextrin (mM) – 0.9 1.9 3.8 7.5 15 15 ized interacting component. Sterol depletion has been previously reported to affect Shh auto- processing10. This depletion was probably somewhat more severe than that produced by our experimental manipulations. Further- c 125 more, we found that Hh signal response is more sensitive than Hh autoprocessing to inhibition by mutational or pharmacological 100 sterol depletion. We therefore conclude that inhibition of response 75 to Hh protein is a more probable cause of the malformations asso- SmoA1 ciated with cholesterol biosynthetic disorders than is inhibition of 50 Hh autoprocessing. Other processes might also be affected by defects in distal cholesterol biosynthesis, as not all of the malforma- 25 tions are necessarily accounted for by impaired Hh signaling. Nev-

relative pathway activity (%) 0 ertheless, our ﬁndings help explain many developmental forskolin 100 µM – + –––––– malformations associated with a relatively common genetic disor-

© Group 2003 Nature Publishing µ compactin 20 M+++++++– der, SLOS, whose incidence among European Caucasians is 1 in cyclodextrin (mM) ––0.9 1.9 3.8 7.5 15 15 22,000 (ref. 19). Our findings may also be relevant to other etiolo- gies of abnormal human development, as holoprosencephaly is Gli family of transcription factors (Fig. 1b). We examined the reported to occur at a frequency of 1 in 250 among aborted ability of sterol perturbations to affect the constitutive Hh path- fetuses20. The surprising connection between cholesterol synthesis way activity in fibroblasts derived from Ptch–/– embryos15,16 and and Hh signal response reported here suggests that signaling path- found a dose-dependent reduction by transient treatment with ways involved in developmental patterning must be considered as increasing concentrations of cyclodextrin in combination with potential targets of any seemingly simple metabolic defect associ- compactin (Fig. 5a). Therefore, sterol depletion can block path- ated with developmental malformations. way activity independently of Ptch action and may act at a point in the pathway downstream of Ptch. Methods We next examined the effects of depleting sterols in NIH3T3 Chick embryos. We applied methyl-β-cyclodextrin (200 µl of a 10% w/v cells stably transfected to express wild-type or oncogenic, acti- solution in L-15 medium (Sigma and Life Technologies)) to windowed, vated Smo (ref. 16; SmoA1; W539L). We found that sterol deple- fertile chick eggs (White Leghorn) after 15 h of incubation. For an average tion could completely block the modest level of pathway egg volume of 50 ml, the final concentration of cyclodextrin was 375 µM. activation produced by overexpression of Smo (Fig. 5b), but that After 4 d of further incubation, we processed the embryos for scanning it scarcely affected pathway activation by SmoA1 (Fig. 5c). The electron microscopy. We dissected the neural plate and epidermal ecto- susceptibility of wild-type Smo but resistance of a mutant acti- derm from stage 9–10 chick embryos, cultured them in collagen and 21 3 β vated form to sterol depletion suggests that Smo may be the site induced them with purified ShhN as described . We added methyl- - cyclodextrin to the chick explant cultures 4 h after induction with ShhN of sterol action. Consistent with this conclusion, sterol depriva- was initiated. tion did not affect the activation of the pathway due to expression of Gli1, which acts downstream of Smo (data not shown). Analysis of Shh protein biogenesis. We plated MEFs in a 10-cm2 dish (Fal- The retention of normal Hh pathway activity in cells expressing con) and transfected them (Fugene 6, Roche) with a full length Shh expres- activated Smo indicates that pathway components downstream sion construct (pRK5-Shh, 5µg) when the cells were roughly 75% conflu- of Smo function normally under conditions of sterol depletion. ent. The next day, we changed the culture medium (Dulbecco’s modified Previous work has suggested that Smo activity is governed by a Eagle medium with 10% fetal bovine serum) to contain either 0.5% fetal balance between active and inactive conformations16. The resistance bovine serum, 0.5% lipid-depleted serum or lipid-depleted serum after a

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30-min treatment with 3.8 mM methyl-β-cyclodextrin (Sigma). After an 3. Cooper, M.K., Porter, J.A., Young, K.E. & Beachy, P.A. Teratogen-mediated additional 24 h in culture, we lysed the MEFs in RIPA buffer (50 mM Tris- inhibition of target tissue response to Shh signaling. Science 280, 1603–1607 (1998). Cl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 4. Chen, J.K., Taipale, J., Cooper, M.K. & Beachy, P.A. Inhibition of Hedgehog 1 mM EDTA, 1 µg ml–1 leupeptin, 1 µg ml aprotinin–1, 0.2 mM phenyl- signaling by direct binding of cyclopamine to Smoothened. Genes Dev. 16, methylsulfonyl fluoride), immunoprecipitated Shh protein from the cell 2743–2748 (2002). 5. Incardona, J.P. et al. Cyclopamine inhibition of Sonic hedgehog signal lysates with a monoclonal antibody that recognizes the N-terminal signal- transduction is not mediated through effects on cholesterol transport. Dev. Biol. ing domain (5E1, Developmental Studies Hybridoma Bank) and 224, 440–452 (2000). immunoblotted them with a polyclonal antibody preparation (JH134). 6. Ohtani, Y., Irie, T., Uekama, K., Fukunaga, K. & Pitha, J. Differential effects of α-, β- and γ-cyclodextrins on human erythrocytes. Eur. J. Biochem. 186, 17–22 (1989). 7. Kilsdonk, E.P. et al. Cellular cholesterol efflux mediated by cyclodextrins. J. Biol. Shh signaling assays. We carried out Shh signaling assays as described16 in Chem. 270, 17250–17256 (1995). primary fibroblasts lacking functional Dhcr7, Sc5d or Ptch and in NIH3T3 8. Chiang, C. et al. Cyclopia and defective axial patterning in mice lacking Sonic –/– +/– hedgehog gene function. Nature 383, 407–413 (1996). fibroblasts. Dhcr7 and Dhcr7 MEFs were generated from embryonic 9. Liem, K.F.Jr., Tremml, G., Roelink, H. & Jessell, T.M. 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